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Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.

Background: The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia)., Methods: We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing., Results: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews., Conclusions: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.

The filaria Brugia beaveri is a parasite of raccoons (Procyon lotor) in Louisiana. Its microfilariae, which circulate in the peripheral blood without any periodicity, develop to the infective stage in mosquitoes. The filaria can be transmitted in the laboratory to other raccoons, the domestic cat, and jirds (Meriones unguiculatus). The prepatent period is 70-107 days depending on the definitive host. Adult worms are found in lymphatics and associated subcutaneous tissues of raccoons and in the heart, lungs, and testes of jirds. In host tissues, the parasite is recognized by its small diameter and the morphology of the body wall. There is a thin cuticle, which is characteristically thickened in the lateral fields; in males, a lateral, internal cuticular ridge is sometimes present. The hypodermis forms large lateral chords and less conspicuous dorsal and ventral chords. Muscle cells are coelomyarian; in females there is an average of 4 cells per body quadrant and in males about 4-6. Internal organs are easily identified as to type, but do not provide any clues to species identification.

Burgia malayi and B. pahangi microfilariae were isolated from the blood of infected Mastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

The cuticle structure of some nematode species was studied by immunogold and lectin-gold techniques. The gold labelling made it possible to distinguish the cuticle layers by the distribution and/or the density of the marker. On the other hand, no labelling pattern was found which led to a clear grouping of the layers into larger 'zones', since there were no subunits consisting of more than one layer which reacted in a characteristic way as compared to the rest of the cuticle. The outer surface of the epicuticle of parasitic adult worms turned out to be highly inert; it did not react with any of the antibodies or lectins tested. The cuticle of parasitic nematodes seems to function as a protection against the host's defense mechanisms rather than as an interaction site. An immunogenic component on the surface was only found in infective larvae. All antibodies and lectins showed a preferential binding to the electron dense layers and fibrous structures (HPL/GalNAc, WGA/GlcNAc) or to the amorphous ground-substance (Con A/Glc, RCA I/Gal).

The larvae of Brugia pahangi recovered from the jird have been shown to grow continuously between moults, their length increasing at least 5-fold during the 4th instar. This intermoult growth is achieved without an increase in the surface area of larvae. The cuticle of very early 4th and 5th-stage worms is highly folded and intermoult growth is accommodated by an expansion of surface annular folds. The major increases in the surface area of larvae occur only through ecdysis, with an 8-fold increase in the surface area being achieved at the 3rd moult. The growth curve of these worms is therefore sigmoidal but the surface area curve is stepped. The relationship between the cuticles of nematodes and insects is discussed.

Brugia timori sp. n. from experimentally-infected Mongolian jirds (Meriones unguiculatus) is described. The adult male differs from other Brugia species, except Brugia malayi, in having a spicular ratio of 3 : 1; it differs from B. malayi in having greater numbers of subventral adanal papillae (up to 5 on each side) that are loosely spaced and irregularly positioned about the cloaca, a greater diameter of the capitulum of the left spicule, greater lengths of the proximal- and midsections of the left spicule, and a greater length of the proximal section of the right spicule. The adult female has an ovejector of greater length and width than that of B. malayi. Microfilariae in the blood of persons from whom the parasite lines of this study originated were typical of the Timor type, having a greater total body length than other Brugia spp., a length to width cephalic space ratio of about 3 : 1, and a sheath which did not stain when processed in the usual manner with Giemsa.

The ultrastruct of the adult subperiodic Brugia malayi (Brug, 1927) within pulmonary arteries of male jirds (Meriones unguiculatus) was studied by transmission electron microscopy. The cuticle consists of 10 sublayers (2 of which are prominently banded) and a typical outer unit membrane. Evidence is presented showing that the subcuticular region of the lateral chords comprises a functional complex of basal infoldings, multivesicular bodies, and associated mitochondria, which is probably engaged in the exchange of solutes across a permeable cuticle. Microbodies with paired, prominent cores, intracisternal A-particle viruslike bodies, nonstaining glycogen patches, and other structures are also present in the lateral chords. The platymyarian somatic musculature shares some coelomyarian characteristics, e.g., apparent neuromuscular connections and prominent glycogen deposits surrounded by mitochondria and other organelles. The alimentary tract has features typical of many nematodes. The luminal segments of the male and female reproductive tracts and their germinal products, excluding microfilariae, are described. Affinities with related species are discussed.

A 27-year-old white woman from New York City acquired an infection by a Brugia species while she camped in the Amazon basin of Peru. She was infected by at least one adult male worm and one gravid female worm. Both worms were intact and in a lymphatic vessel of a right cervical lymph node. The lymph node and surrounding fibroadipose tissue contained many microfilariae. The male worm was 50 micron wide and the female, 100 micron. Both worms had thin (1 micron) cuticles with fine transverse striations. There were 3 to 4 somatic muscle cells per quadrant. Microfilariae had tails characteristic of the genus Brugia. Although specific identification was not possible from the available material, the worm closely resembled Brugia guyanensis, a parasite of the coatimundi (Nasua nasua) and the only species of Brugia known in South America.

Infective larvae of Brugia pahangi were irradiated at 10, 25 or 45 krads by means of a Cobalt 60 source. In cats, 10 krads caused the worms to be stunted and sterile but allowed them to become 5th stage, migrate posteriorly into the afferent lymphatic, and produce pathology. 25 krads prevented the worms from developing beyond the early fourth stage and from migrating away from the popliteal lymph node. No gross pathological reacions were evident. 45 krads produced the same effects as 25 krads but the longevity of the worms was much reduced.

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK2 rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.

During studies on filariasis in Thailand, attempts were made to differentiate Wuchereria bancrofti and Brugia spp. by morphological characteristics, as well as by vector species.

When jirds were infected with a single inoculum of 25-50 infective larvae of Brugia pahangi an overall mean recovery of adult worms of 44.5% (n = 41) was obtained. There was no difference in recoveries between male and female jirds. If jirds were repeatedly inoculated with larvae into the peritoneal cavity yields were only slightly reduced. Yields were 30.5% for 5 infections (n = 10), 26.7% for 10 infections (n = 8), 34.4% for 15 infections (n = 10) and 28.5% for 20 infections (n = 7). Twice as many worms were recovered from intraperitoneally inoculated jirds than from subcutaneously inoculated jirds.

Nine new zoonotic Brugia infections, 8 acquired in the United States and 1 in Brazil, are described. In each, a single worm was found in histological sections of lymph nodes or lymphoid tissues. In those from North America, 5 of the 8 worms were females, all were infertile, and only 3 were alive at the time the tissues were excised; all of the patients were male. A dead female worm was collected from a Brazilian male patient. In addition, 3 cases of filarial infections in the U.S., previously identified as Dirofilaria-, Dipetalonema-, or Brugia-like, are reviewed and reclassified as Brugia species on the basis of microscopic morphological features. Morphological features of male and female zoonotic Brugia species as they appear in the host tissues are described. The possible reservoirs of human infections are discussed.

The susceptibility of Mongolian jirds, Meriones unguiculatus, and PD4 hamsters, Mesocricetus auratus, to Brugia pahangi was compared based on the percentage adult worm recoveries, mean microfilaremia levels, and adult worm lengths. Fourteen male jirds and seventeen male PD4 hamsters were each inoculated subcutaneously in the left inguinal region with 90-100 L3 of B. pahangi and necropsied 130-150 days after inoculation. There were no significant differences between jirds and hamsters in mean adult worm recoveries (24.7 vs 25.4%) and prepatent periods (69.9 vs 77 days after inoculation). In hamsters, 85% of recovered worms were found in the heart and lungs and 15% were found in genital lymphatic vessels. In jirds, distribution of recovered worms was 66% in genital lymphatics, 23% in the heart and lungs, 8% in the peritoneal cavity, and 3% in lymphatic vessels in other sites. The mean microfilaremia level in jirds (16.5/20 microliter) was significantly higher than in hamsters (8.7/20 microliter. Female worms in the genital lymphatics of jirds were significantly longer than female worms in the genital lymphatics of PD4 hamsters (33.5 vs 27.3 mm). Lengths of worms in other locations were similar between the two species.

Two male and two female Brugia malayi were collected from Mastomys natalensis, and cross sections from various regions of these worms were studied. The body wall of the female worm is thin throughout its length, whereas in the midbody and posterior region of the male worm the somatic muscles form a thick layer. In both sexes the intestine is thickest immediately behind the oesophagus and tapers posteriorly. The lumen was empty in all sections. In both genital tubes of the female worm a successive row of developmental stages could be observed. The rather short ovaries lie in the posterior region. The fertilisation chambers and the following regions are the thickest portions of the uteri. The testis is situated in the anterior region of the male worm, whereas in the midbody and posterior regions the vas deferens contains stretched spermatozoa. Compared to other filariae the sexual dimorphism is not very pronounced in this species.

Scanning Electron Microscopic (SEM) observations were made on the adult worms of Brugia malayi from rodent hosts. Descriptions of the surface features of mouth, vulva, anus, caudal papillae and spicules are given.

Brugia lepori sp. n., a filarial nematode from the abdominal lymphatics and subcutaneous tissues of rabbits (Sylvilagus aquaticus, S. floridanus), from St. Tammany Parish, Louisiana, is described. Brugia lepori is of moderate size (males 12 to 19 mm, females 39 to 45 mm) and within the genus most closely resembles Brugia beaveri of the raccoon, from which it can be distinguished by its larger size, smaller spicules, and smaller microfilaria which has a shorter cephalic space. Brugia lepori is only the second species of Brugia described from North America and the third species reported from the Western Hemisphere.

A case of human brugiasis in a student from Gambela, Ethiopia, is reported. Ten sheathed microfilariae showing the Brugia genus characteristics were recovered from 1 ml of blood.

The diagnostic features in tissue sections from patients with zoonotic filarial infections are reviewed. In general, two types of infections are recognized: 1) those presenting radiologically in healthy patients as coin lesions, which are usually removed because a clinical diagnosis of carcinoma of the lung is made and infarcts containing Dirofilaria immitis parasites in pulmonary arteries are found; and 2) those presenting as subcutaneous nodules, in which D. tenuis, a parasite from raccoons, D. repens, from dogs and cats, D. ursi , from bears, and Onchocerca spp, from horses or cattle, require delineation. Moreover, nodules that on microscopic examination are seen to be lymph nodes may harbor Brugia spp. The geographic distribution of these infections, with emphasis on the United States, is discussed.